Protein Prenylation PART A

340 Pages·2011·16.828 MB·1-343\340

by  Fuyuhiko Tamanoi, Christine A. Hrycyna and Martin O. Bergo (Eds.)

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Preface
The posttranslational modification of eukaryotic proteins by the addi-
tionofisoprenyllipidsattheirC-terminiwasfirstobservedinthe1970sand
1980s.Thesediscoveriesemergedfromtwodifferentlinesofinvestigation.
First,studiesofcellstreatedwithcholesterolbiosynthesisinhibitorsledto
theidentificationofprenylatedproteinssuchasthenuclearlamins.Second,
studiesonyeasts,suchasRhodosporidiumtoluroides,revealedthepresence
of isoprenoids in peptide mating pheromones. Since then, more than a
hundredproteinshavebeenshowntobe modified byC15 farnesyl orC20
geranylgeranylgroups,includingmostofthemembersoftheRas,Rho,and
Rab families of G proteins. The precise number of prenylated proteins is
not yet known, but novel proteomics approaches have been developed to
definetheentireprenylatedproteome.
Soon after these modifications were discovered, the search for prenyl-
transferaseenzymesintensified.Theenzymesthatcatalyzeproteinprenyla-
tion,includingproteinfarnesyltransferase,proteingeranylgeranyltransferase
type I, and protein geranylgeranyltransferase type II (Rab geranylgeranyl-
transferase) were identified and characterized in the 1990s. Subsequent
structural studies identified subunit composition and catalytic properties
of these enzymes and resulted in a more complete understanding of the
mechanismofactionattheatomiclevel.
In the late 1980s, it became clear that for some proteins that contain a
C-terminal CaaX sequence (C is cysteine, ‘‘a’’ is a generally aliphatic
residue,andXisoneofanumberofaminoacids),prenylationisfollowed
by proteolytic cleavage of C-terminal aaX residues and carboxylmethyla-
tionofthenewlyexposedC-terminalcysteine.Theenzymesresponsiblefor
these modifications, Rce1 and Icmt, respectively, were subsequently iden-
tifiedandtheirbiologicalandbiochemicalcharacterizationisongoing.
Since the discovery of these modifications, the biological significance of
proteinprenylationandthesubsequentmodificationshavebeeninvestigated
extensively. The modifications are thought to be important for membrane
association, protein–protein interactions, protein stability, and receptor–
ligand interactions. In particular, the demonstration that the transforming
activity of mutant Ras depends on prenylation and methylation pointed to
the significance of these modifications in oncogenesis. More recently,
xi
xii PREFACE
importantinsightsintothe biologicalroleofthese modificationshave been
obtainedbyaseriesofstudiesusingknockoutmice.
Finally, small molecule inhibitors of protein farnesyltransferase (FTIs),
protein geranylgeranyltransferase (GGTIs), and Rabgeranylgeranyltrans-
ferasehavebeendevelopedandbothFTIsandGGTIshavebeenevaluated
asanticancerdrugs.Inaddition,smallmoleculeinhibitorsofRce1andIcmt
havebeendeveloped.
To encompass past and recent developments in the study of protein
prenylation, we designed a two-part miniseries. This volume is the first
part (Volume 29, Part A). We believe that this miniseries captures the
current knowledge and advances inthe field and hope that they will be of
interesttoawiderangeofresearchersincludingbiochemists,molecularand
cellbiologists,andcancerresearchers.
Wethanktheauthorsfortheirexcellentandinformativechapters,Mary
AnnZimmermanandMalathiSamayanofElsevierfortheirguidance,and
GloriaLeeatUCLAforheroutstandingassistanceincommunicationand
inpreparingandeditingchapters.
FuyuhikoTamanoi
ChristineA.Hrycyna
MartinO.Bergo
May2011
1
Protein Prenylation: A Perspective on
Initial Discoveries
JOHNA.GLOMSET
DepartmentsofMedicineandBiochemistryandRegionalPrimateResearchCenter
UniversityofWashington,Seattle
Washington,USA
I. Abstract
The discovery of protein prenylation by members of our group was the
indirect result of our early studies of the effects of plasma lipoproteins on
arterial smooth muscle cells. We had discovered that platelets contain
PDGF,agrowthfactorthatcanstimulatethereplicationofcellsinculture,
and had begun to use partially purified PDGF to examine the effects of
cholesterol on different types of replicating cells. For example, we added
highconcentrationsofcompactin(aninhibitorof3-hydroxy-3-methylglutaryl
coenzyme A reductase) to the cells to block the synthesis of cholesterol,
showedthatthecompactinalsoblockedthesynthesisofDNAandinduced
cell rounding, and sought to determine whether added unesterified choles-
terolorplasmalipoproteinscouldpreventtheseeffects.However,neitherof
these additives was effective, while added exogenous mevalonic acid pre-
vented both effects.So weadded radioactive mevalonic acid tocompactin-
treatedcellsinamountsthatcouldpromoteshapechangereversal,searched
forproductsofthemevalonicacidthatcouldhavebeeninvolved,andfound
thatmuchoftheradioactivitywasinsolubleinlipidsolvents.Further,astudy
of Swiss 3T3 cells that had been cultured in the presence of mevinolin (an
inhibitor of mevalonic acid synthesis) and then labeled with radioactive
exogenous mevalonic acid showed that much of the material that became
THEENZYMES,Vol.XXIX 1 ISSNNO:1874-6047
#2011ElsevierInc.Allrightsreserved. DOI:10.1016/B978-0-12-381339-8.00001-9
2 JOHNA.GLOMSET
labeled was present in proteins which had apparent molecular masses of
13,000–58,000Da. And subsequent studies of other cells identified specific
proteinsthatweremodifiedbyfarnesylgroupsorgeranylgeranylgroups.
II. StepsintheTrailofResearch
Manyyearsago,RussellRossandIdecidedtodoacollaborativestudyof
theeffectsofplasmalipoproteinsonculturesofarterialsmoothmusclecells,
and to use plasma lipoproteins and arterial smooth muscle cells from non-
human primates as models. So, I obtained blood from caged nonhuman
primates in the Regional Primate Research Center at the University of
Washington and used a centrifugation approach to separate the plasma
lipoproteins from the blood cells, while members of the Ross laboratory
obtained fresh arterial tissue from the Primate Center’s tissue distribution
program,andusedthistissuetoprepareculturesofthearterialtissuesmooth
musclecells.However,whenthelipoproteinsthatIhadisolatedwereadded
to the cultures of smooth muscle cells, the smooth muscle cells failed to
replicate, and we later discovered that the reason for this was that the
centrifugationprocedurewhichIhadusedtopreparetheplasmalipoproteins
had effectively separated the lipoproteins from platelets and a previously
unknowngrowthfactorthattheycontained[1].Further,subsequentexperi-
ments showed that a partially purified form of this growth factor (PDGF)
could not only stimulate the replication of arterial smooth muscle cells in
culturebutalsostimulatethesynthesisofDNAandcelldivisioninSwiss3T3
cellsinculture[2,3].
We then studied cultures of PDGF-stimulated nonhuman primate
arterial smooth muscle cells or Swiss 3T3 cells to investigate the relation
betweencholesterolsynthesisandDNAsynthesiswithinasinglecellcycle.
In each case, we used high concentrations of compactin (ML-236B), an
inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, to block
the synthesis of cholesterol, examined the effect of this block on the
synthesis of DNA, and then sought to determine whether added exoge-
nous cholesterol could prevent the effect of the compactin on DNA
synthesis. The results of these experiments showed that compactin could
indeed block the synthesis of DNA in each type of cell, but we were
unable to prevent or reverse this effect by adding exogenous unesterified
cholesterol or low- density lipoproteins to the cells. However, we found
that we could prevent the effect of compactin on DNA synthesis by
adding exogenous mevalonic acid to the cells, and this raised the possibil-
ity that either mevalonic acid itself or an unknown product of mevalonic
1. APERSPECTIVEONINITIALDISCOVERIES 3
acid metabolism might have promoted the growth of the cells. Note that
other investigators had been considering this possibility as well [4,5].
WenextexaminedtheeffectsofcompactinonSwiss3T3cellsthatwere
eitherquiescentortraversingthroughasinglecellcycleinresponsetoadded
PDGF, and found that compactin could not only inhibit the synthesis of
DNAinthePDGF-stimulatedcellsbutalsocauseboththequiescentcells
and the replicating cells to acquire a characteristic rounded shape [6]. To
explorethemolecularbasisoftheseeffects,weaddedradioactivemevalonic
acid to cultures of Swiss 3T3 cells and sought to identify a product of
mevalonic acid metabolism that could promote the synthesis of DNA and
regulate the shape of the cells. The initial results of these experiments
showedthatthecellsincorporatedmuchoftheradioactivemevalonicacid
into cell proteins [7], and subsequent studies showed that a few specific
proteinswereinvolved[8].Forexample,studiesofHeLacellsandChinese
hamsterovarycellsprovidedevidencethatthelaminBinthesecellscanbe
modified by a derivative of mevalonic acid [9] and that the corresponding
derivativeinhumanHeLacellsisafarnesylgroupwhichmodifiesacysteine
residuetowardtheendoflaminB[10].Moreover,astudyofSaccharomyces
cerevisiae identified mutants that were defective in Ras protein farnesyla-
tionandshowedthatatleasttwogeneswereinvolved[11].
Inadditiontothis,astudyofHeLacellsthathadbeenincubatedinthe
presenceofradioactivemevalonicacididentifiedlabeledfragmentsofcell
proteins that contained an all-trans geranylgeranyl group [12]. Brain G
protein gamma subunits were found to contain an all-trans geranylgera-
nyl-cysteine methyl ester at their carboxyl termini [13]. Evidence was
obtained that the human platelet protein, smg p21B is geranylgeranylated
andcarboxylmethylatedatitsC-terminalcysteineresidue[14].Theproper-
tiesofaproteingeranylgeranyltransferasefrombovinebrainwerestudied
[15]. The C-terminus of smg p25A was shown to contain two geranylger-
anylated cysteine residues and a methyl ester [16]. A Rab geranylgeranyl
transferase was shown to catalyze the geranylgeranylation of adjacent
cysteines in the small GTPases Rab1A, Rab3A, and Rab5A [17]. And
several reviews related to protein prenylation were published including
onethatdescribedthemethodologythatwehadbeenusingtocharacterize
protein-boundprenylgroups[18–20].
REFERENCES
1. Ross,R.,Glomset,J.A.,Kariya,B.,andHarker,L.(1974).Aplatelet-dependentserum
factorthatstimulatestheproliferationofarterialsmoothmusclecellsinvitro.ProcNatl
AcadSciUSA71:1207–1210.
2. Rutherford,R.B.,andRoss,R.(1976).Plateletfactorsstimulatefibroblastsandsmooth
musclecellsquiescentinplasmaserumtoproliferate.JCellBiol69:196–203.
4 JOHNA.GLOMSET
3. Vogel,A.,Raines,E.,Kariya,B.,Rivest,M.J.,andRoss,R.(1978).Coordinatecontrolof
3T3 cell proliferation by platelet-derived growth factor and plasma components. Proc
NatlAcadSciUSA75:2810–2814.
4. Kaneko,I.,Hazama-Shimada,Y.,andEndo,A.(1978).Inhibitoryeffectsonlipidmetab-
olism in cultured cells of ML-236B, a potent inhibitor of 3-hydroxy-3-methylglutaryl-
coenzyme-Areductase.EurJBiochem87:313–321.
5. Quesney-Huneeus, V., Wiley, M.H., and Siperstein, M.D. (1979). Essential role for
mevalonatesynthesisinDNAreplication.ProcNatlAcadSciUSA76:5056–5060.
6. Habenicht,A.J.,Glomset,J.A.,andRoss,R.(1980).Relationofcholesterolandmeva-
lonicacidtothecellcycleinsmoothmuscleandswiss3T3cellsstimulatedtodivideby
platelet-derivedgrowthfactor.JBiolChem255:5134–5140.
7. Schmidt,R.A.,Glomset,J.A.,Wight,T.N.,Habenicht,A.J.,andRoss,R.(1982).Astudy
oftheInfluenceofmevalonicacidanditsmetabolitesonthemorphologyofswiss3T3
cells.JCellBiol95:144–153.
8. Schmidt,R.A.,Schneider,C.J.,andGlomset,J.A.(1984).Evidenceforpost-translational
incorporationofaproductofmevalonicacidintoSwiss3T3cellproteins.JBiolChem
259:10175–10180.
9. Wolda,S.L.,andGlomset,J.A.(1988).EvidenceformodificationoflaminBbyaproduct
ofmevalonicacid.JBiolChem263:5997–6000.
10. Farnsworth,C.C.,Wolda,S.L.,Gelb,M.H.,andGlomset,J.A.(1989).HumanlaminB
containsafarnesylatedcysteineresidue.JBiolChem264:20422–20429.
11. Goodman, L.E., Judd, S.R., Farnsworth, C.C., Powers, S., and Gelb, M.H. (1990).
Mutants of Saccharomyces cerevisiae defective in the farnesylation of Ras proteins.
ProcNatlAcadSciUSA87:9665–9669.
12. Farnsworth,C.C.,Gelb,M.H.,andGlomset,J.A.(1990).Identificationofgeranylgeranyl-
modifiedproteinsinHeLacells.Science247:320–322.
13. Yamane,H.K.,Farnsworth,C.C.,Xie,H.,Howald,W.,Fung,B.K.K.,Clarke,S.,Gelb,M.H.,
and Glomset, J.A. (1990). Brain G protein gamma subunits contain an all-trans-
geranylgeranylcysteinemethylesterattheircarboxyltermini.ProcNatlAcadSciUSA
87:5868–5872.
14. Kawata,M.,Farnsworth,C.C.,Yoshida,Y.,Gelb,M.H.,Glomset,J.A.,andTakai,Y.
(1990).Posttranslationallyprocessedstructureofthehumanplateletproteinsmgp21B:
evidenceforgeranylgeranylationandcarboxylmethylationoftheC-terminalcysteine.
ProcNatlAcadSciUSA87:8960–8964.
15. Yokoyama,K.,Goodwin,G.W.,Ghomashchi,F.,Glomset,J.A.,andGelb,M.H.(1991).
Aproteingeranylgeranyltransferasefrombovinebrain:implicationsforproteinprenyla-
tionspecificity.ProcNatlAcadSciUSA88:5302–5306.
16. Farnsworth,C.C.,Kawata,M.,Yoshida,Y.,Takai,Y.,Gelb,M.H.,andGlomset,J.A.
(1991).CterminusofthesmallGTP-bindingproteinsmgp25Acontainstwogeranylger-
anylatedcysteineresiduesandamethylester.ProcNatlAcadSciUSA88:6196–6200.
17. Farnsworth,C.C.,Seabra,M.C.,Ericsson,L.H.,Gelb,M.H.,andGlomset,J.A.(1994).Rab
geranylgeranyltransferasecatalyzesthegeranylgeranylationofadjacentcysteinesinthe
smallGTPasesRab1A,Rab3A,andRab5A.ProcNatlAcadSciUSA91:11963–11967.
18. Glomset,J.A.,Gelb,M.H.,andFarnsworth,C.C.(1990).Prenylproteinsineukaryotic
cells:anewtypeofmembraneanchor.TrendsBiochemSci15:139–142.
19. Farnsworth, C.C., Casey, P.J., Howald, W.N., Glomset, J.A., and Gelb, M.H. (1990).
Structuralcharacterizationofprenylgroupsattachedtoproteins.Methods1:231–240.
20. Glomset,J.A.,andFarnsworth,C.C.(1994).Roleofproteinmodificationreactionsin
programminginteractionsbetweenras-relatedGTPasesandcellmembranes.AnnuRev
CellBiol10:181–205.
2
Insights into the Function of
Prenylation from Nuclear Lamin
Farnesylation
MICHAELSINENSKY
DepartmentofBiochemistryandMolecularBiology
EastTennesseeStateUniversity,JohnsonCity
Tennessee,USA
I. Abstract
The discovery of mammalian protein prenylation was originally moti-
vatedbyanefforttoidentifyanonsterolisoprenoidwhichindirectevidence
suggestedwasacoregulatorofisoprenoidbiosynthesisandplayedacritical
role in cellular proliferation. The first prenylated proteins to be identified
were the nuclear lamin proteins—B lamins and prelamin A—which were
subsequentlyshowntobefarnesylatedatacarboxyl-terminalCAAXmotif.
Inbothtypesoflamin,thefarnesylationandcarboxymethylationplayarole
in targeting these proteins to the nuclear envelope. The nucleus can be
demonstratedtobeaCAAXprocessingcompartmentforthelamins.Inthe
caseofprelaminA,thereisremovalofacarboxyl-terminalpolypeptidewhich
is specifically catalyzed by the enzyme Zmpste24. This processing event is
necessaryforassemblyoflaminAintothelaminaandmayplayaroleincell
cyclecontrol.Becausethenucleuscontainsonlyonetargetmembrane,lamin
farnesylationandcarboxymethylationmaybesufficienttoallowassociation
withthismembrane.Thisstandsincontrasttofarnesylatedproteinsexpressed
inthecytoplasm.
THEENZYMES,Vol.XXIX 5 ISSNNO:1874-6047
#2011ElsevierInc.Allrightsreserved. DOI:10.1016/B978-0-12-381339-8.00002-0
6 MICHAELSINENSKY
II. Introduction
Thediscoveryofproteinprenylation,asageneralphenomenon,andthe
more particular discovery of lamin farnesylation began with studies
directed at understanding the regulation of isoprenoid metabolism. Early
effortstounderstandthe regulation ofcholesterolbiosynthesishad by the
late 1970s led to the confirmation of the hypothesis that regulation was
centered on the enzyme 3-hydroxy-3-methyl glutaryl coenzyme A reduc-
tase (HMGR). Driven by the importance of cholesterol metabolism in
atherosclerosis, pharmaceutical research directed at the discovery of
drugs useful in lowering serum cholesterol levels became focused on the
searchfordrugsthatcouldinhibitHMGR.Themostsuccessfulclassofsuch
compounds are the statins (e.g., lovastatin), the first of which, compactin,
wasreportedbyEndoin1976[1].
SubsequentstudieswithcompactinontheregulationofHMGRledtothe
conceptthatnotonlywasHMGRregulatedinresponsetosterolsbut,asthe
rate limiting step of isoprenoid biosynthesis, was also regulated by other
isoprenoids(reviewedinRef.[2]).Thiswasmostclearlyexhibitedasaloss
ofthesterolregulatoryresponseofHMGRincellsstarvedformevalonateby
compactin treatment. Other studies on compactin effects on the cycling of
culturedcellsrevealedthatamevalonatemetabolite,otherthancholesterol,
wasrequiredforreentryofquiescentcellsintoS-phase[3,4].Thisobservation
suggestedthatitwouldbepossibletoisolateasomaticcellmutantauxotro-
phic for mevalonate via the bromodeoxyuridine (BrdU)-visible light tech-
nique.Thisprocedureselectsformutantsthatundergoacellcyclearrestin
responsetostarvationforarequirednutrientpreventingtheincorporationof
light-activatedtoxicBrdUintoDNA[5].Theresultantmevalonateauxotroph
(Mev-1)wasexpectedtobedefectiveinoneoftheenzymesofmevalonate
synthesiswhichturnedouttobecytosolic3-hydroxy-3-methylglutarylcoen-
zymeAsynthasebasedonenzymeassay[6,7].Aswasthecasewithcultured
cellstreatedwithcompactin,Mev-1exhibiteddefectiveregulationofHMGR
bysterolsduringmevalonate starvation.These observations weregenerally
takenasconsistentwiththeviewthattherewasanonsterolisoprenoidcritical
forcellcycleprogressionand,hence,alsoregulatoryformevalonatebiosyn-
thesis—aconceptdubbed‘‘multivalentregulation’’[2].
A. DISCOVERYOFLAMINFARNESYLATION
The discovery by the Glomset laboratory [8] of the posttranslational
incorporation of labeled mevalonate into proteins in cultured mammalian
cells suggested the hypothesis that a protein modified with an isoprenoid
2. INSIGHTSINTOTHEFUNCTIONOFPRENYLATION 7
substituent was this coregulatory molecule. This hypothesis was shown to
be supported by the kinetic relationship between isoprenylated protein
turnover and cell cycle arrest in mevalonate starved Mev-1 cells [9].
At this point, there had not yet been any identification of a mammalian
protein that was prenylated, a metabolic process that (as discussed in
Volume 30 Protein Prenylation Part B and in [10]) was known only for
fungi. To explore for a possible candidate protein, it seemed plausible to
examinemammaliancellnucleiforproteinswhichincorporatedlabelfrom
mevalonate in the hope that whatever protein(s) might be involved in
cell cycle control were affecting DNA synthesis directly. Exploration of
mevalonate-labelednuclearproteins[11]ledtothefindingthattherewere
threesuchproteinswhich,eventually,couldbedemonstratedtobelaminB
[11,12],prelaminA[11,13],andlaminB (unpublishedresults).Mevalonate
2
incorporation into the respective lamin proteins can most readily be illu-
strated by 2D-NEPHGE electrophoresis of nuclear matrix intermediate
filament preparations [13,14] (Figure 2.1). Verification of the identity of
laminB,asindicatedby2D-gel migration,was performed byimmunopre-
cipitation and was initially reported simultaneously by our laboratory and
thatofGlomset[12].Theidentityoftheprenylgroupasfarnesylwaslater
reportedbytheGlomsetlabaswell[15],andlaminBthusbecamethefirst
exampleofaprenylatedmammalianprotein.
Ourlaboratorychosetopursuethebiochemistryandcellphysiologyof
prelamin A prenylation. This was motivated by our original interest in a
possible role for one or more prenylated proteins in cell cycle regulation.
A B
Ao B2 Ao
B B
2 A A
B
C
v B:lamin B; B2:lamin B2;
A: lamin A;C: lamin C;
A : prelamin A
o
a:actin;v:vimentin
a
FIG. 2.1. Coomassie stained (A) or R-[2-14C] mevalonate-labeled proteins (B) found in
nuclearmatrixintermediatefilamentpreparationsfromculturedCHOcells.Suchpreparations
[13]areenrichedfornuclearlaminproteins.Radioactivityisvisualizedbyfluorography.The
pHincreasesfromlefttoright.